RESUMEN
Glucitol, also known as sorbitol, is a major photosynthetic product in plants from the Rosaceae family. This sugar alcohol is synthesized from glucose-6-phosphate by the combined activities of aldose-6-phosphate reductase (Ald6PRase) and glucitol-6-phosphatase. In this work we show the purification and characterization of recombinant Ald6PRase from peach leaves. The recombinant enzyme was inhibited by glucose-1-phosphate, fructose-6-phosphate, fructose-1,6-bisphosphate and orthophosphate. Oxidizing agents irreversibly inhibited the enzyme and produced protein precipitation. Enzyme thiolation with oxidized glutathione protected the enzyme from insolubilization caused by diamide, while incubation with NADP+ (one of the substrates) completely prevented enzyme precipitation. Our results suggest that Ald6PRase is finely regulated to control carbon partitioning in peach leaves.
Asunto(s)
Aldehído Reductasa/metabolismo , Hojas de la Planta/enzimología , Proteínas de Plantas/metabolismo , Prunus domestica/enzimología , Aldehído Reductasa/antagonistas & inhibidores , Aldehído Reductasa/genética , Fructosadifosfatos/metabolismo , Fructosadifosfatos/farmacología , Fructosafosfatos/metabolismo , Fructosafosfatos/farmacología , Glucofosfatos/metabolismo , Glucofosfatos/farmacología , Disulfuro de Glutatión/metabolismo , Hexosafosfatos/metabolismo , Hexosafosfatos/farmacología , Immunoblotting , Cinética , Modelos Biológicos , NADP/metabolismo , Oxidantes/metabolismo , Oxidantes/farmacología , Fosfatos/metabolismo , Fosfatos/farmacología , Filogenia , Hojas de la Planta/genética , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Prunus domestica/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Compuestos de Sulfhidrilo/metabolismoRESUMEN
Assay conditions have been established which allow maximum expression of phosphodeoxyribomutase activity in ultrasonic extracts of Escherichia coli K-12. The enzyme requires 2-mercaptoethanol, manganous ions, and glucose 1,6-diphosphate for optimal activity. When cells are grown in minimal medium with glycerol as the carbon source and supplemented with 10(-3)m thymidine, phosphodeoxyribomutase is induced three- to four-fold. Cell pellets may be frozen for 24 hr before sonic disruption without loss of enzyme activity. The assay is highly reproducible.